Microbiological studies of plaque-biofilms from toddlers reveal an association between early-childhood caries (ECC) and the presence of C. albicans, along with elevated populations of S. mutans. How this association is implicated in the pathogenesis of the disease remains an enigma. Our preliminary data provide striking evidence that S. mutans and C. albicans develop a symbiotic relationship that synergizes virulence of plaque- biofilms in the presence of sucrose, amplifying the severity of carious lesions in vivo. Using our rodent model of ECC, we observed enhanced levels of infection with elevated carriage of both S. mutans and C. albicans within plaque-biofilms from co-infected animals compared to those infected with either species alone. Importantly, the virulence of plaque-biofilm in co-infected animal was synergistically enhanced, leading to the development of rampant carious lesions on smooth-surface of teeth (similar to those found in ECC). The observed synergism is fascinating because C. albicans usually does not associate well with S. mutans. However, further in vitro studies revealed that S. mutans-derived exoenzyme termed GtfB, acting with sucrose, plays a central role in the ability of S. mutans and C. albicans to associate with each other and form co-species biofilms. As co- species biofilm is initiated, the presence of C. albicans dramatically modifies the physical environment of the biofilm by enhancing the assembly of highly insoluble and diffusion-limiting EPS matrix and the growth of S. mutans microcolonies. Thus, C. albicans-derived factors contribute with co-species biofilm development. We hypothesize that S. mutans-C.albicans association mediated via GtfB and C. albicans factors modulate the development of hypercariogenic biofilms on teeth. It is conceivable that the alterations in the extracellular matrix containing a dense population of acidogenic microorganisms enhance acid accumulation locally, which is critical for the development of carious lesions. To test our hypothesis, we will focus on three aims: In Aim 1, we will identify C. albicans mutants defective in the ability to develop co-species biofilm with S. mutans using unbiased genetic approach (by screening an available library of transcription factor deletion mutants) combined with biochemical and confocal microscopy methods in vitro. The C. albicans defective in co-species biofilm and gtfB-defective S. mutans (and their parental strains) will be then examined for their influence on biofilm pH microenvironment in vitro (Aim 2) and expression of biofilm virulence in vivo (Aim 3). In Aim 2, we will investigate how bacterial-fungal interactions modulate the spatio-temporal development of acidic niches within intact biofilm architecture using our time-lapsed pH mapping. Lastly, in Aim 3, we will examine the role of GtfB and C. albicans factors in the pathogenesis of dental caries in vivo using C. albicans defective in co-species biofilm and gtfB-defective S. mutans (with their parental strains as controls) with our rodent model. A comprehensive program from laboratory studies to in vivo investigations is offered to provide critical insights into the molecular mechanisms of this highly virulent cross-kingdom interaction and their implications in ECC.